Human embryonic stem (ES) cells are pluripotent cells isolated from developing blastocysts. Induced pluripotent stem cells (iPS cells) are pluripotent cells originally isolated from somatic cells of the body reprogrammed by genetic and non-genetic approaches (for review see Amabile and Meissner, 2009). While iPS cells share many characteristics of ES cells including the ability to be differentiated in vitro to cells of all three germ layers, they are not identical. Genetic and epigenetic differences between these two cell types have been reported in the literature and these differences may contribute to altered differentiation efficiencies when subjected to in vitro differentiation protocols. ES cells as well as iPS cells serve as an excellent in vitro system for studying differentiation events and as unlimited source for generating various specialized cell types in large quantities for basic research, drug screening and regenerative therapeutic applications.
Protocols to induce a certain germ layer cell type and subsequent definitive tissue types from human pluripotent stem cells, which includes human embryonic stem cells and induced pluripotent stem cells, are numerous, diverse and currently not standardized. They commonly involve differentiation using 3 major categories of protocols (for a review see Murry and Keller, 2008):                1. Based on co-culture of pluripotent stem cells with other cell types such as feeder cells (e.g. D'Amour et al., 2005, Perrier et al., 2005) or a somatic cell type (eg. induction of cardiomyocytes via co-culture with murine endoderm-like cell lines (Mummery et al., 2007); in medium conditioned by the feeder cells, which induces a certain germ layer fate (Schulz et al., 2003) or alternatively with the addition of factors (D'Amour et al., 2005);        2. Based on adherent culture as monolayers with or in the absence of serum and including the addition of morphogens (Nat et al., 2007; Chambers et al., 2009). Monolayer cultures of pluripotent stem cells can be achieved by dissociating pluripotent stem cells to single cells and then plating those single cells on a coated or uncoated culture surface. Typically, pluripotent stem cells are plated onto culture surfaces that have been precoated with extracellular matrix proteins or synthetic peptides that promote the attachment and survival of pluripotent stem cells. Common proteins and peptides that support this attachment and survival are generally known to those in the field and include for example Matrigel™, vitronectin, E-Cadherin, and laminin. Synthetic peptides that can serve as substrates for pluripotent stem cells are also known to those in the field and can include for example integrin-binding RGD peptides. Additionally, cells can be seeded onto synthetic or biological scaffolds, including artificial organ scaffolds or de-cellularized organs or tissues. Generally, single cells are plated at a known density yielding a monolayer of a defined confluence. There is evidence that cell plating densities can affect cultured cells by influencing cell growth, death, and differentiation. For example, plating efficiency of human pluripotent stem cells is improved if single cells are plated at higher density or if the cells are maintained as clumps where localized areas of high density can improve cell survival. Many protocols for the differentiation of pluripotent stem cells to more specified lineages require plating of pluripotent stem cells at a particular confluence. Confluence is typically assessed by the user by visually assessing the percentage of the culture surface covered by the adherent cells. For example, a 50% confluent culture would appear to have adhered cells covering half of the area of the culture surface. A 100% confluent culture would appear to have cells covering the entire culture surface. Confluence therefore does not indicate a particular number of cells given that cells can be of different size (the same number of smaller cells will cover less area than larger cells) or may spread out on a culture surface to different degrees. Pluripotent stem cells can also be plated as clumps or aggregates of 2 or more cells adhered to each other. These clumps also require similar attachment substrates to those required for single pluripotent stem cells. These clumps are typically multilayered and confluence of these cultures is assessed again by estimating the percentage of the culture surface that is not covered by these adhered clumps. Cultures can also be stacked on top of one another to create multilayered or 3-dimensional cultures. In this type of culture system, cells from one monolayer can either be directly in contact with the adjacent monolayer, or the monolayers can be separated from each other by a matrix or other biological or physical barrier;        3. Based on the formation of 3-D aggregates called embryoid bodies (EBs). Cells in the EBs are multipotential, with the propensity to develop into cells of any of the 3 germ layers (endoderm, mesoderm or ectoderm) (Odorico et al., 2001). Usually morphogens are also added either directly at the time of EB formation to serve as inductive cues or at a later time-point (e.g. after plating of the EBs) to selectively support survival of or differentiation to the desired cell lineage (see embryonic stem cell protocols). EBs can be cultured in suspension, for example in ultra-low adherent culture plates or in bioreactors, or they can re-adhered to a culture surface.        
The major disadvantages of these protocols can be summarized as follows. Exact protocol details can vary significantly between different labs as well as different operators within a single lab. In addition, protocol efficiencies can vary among different cell lines and within the same cell line if cultured using alternate methods. Thus reproducing published or disclosed protocols can require significant optimization. Media formulations used in the 3 different categories of protocols above consist of a variety of media components, additives, and supplement mixtures that are not consistent between labs and protocols. Detailed lists of the individual components in culture medium and their working concentrations are very often not available especially when pre-mixed supplements from commercial vendors are used.
The cultures derived from pluripotent stem cells even under the most defined conditions, are inherently heterogeneous, consisting of cell types of different lineages and at different stages of development. Heterogeneity may be explained by intrinsic cell-to-cell signaling and the variations in the time points used when manipulating the cells in some of the protocols. One solution that has been applied to increase the percentages of the desired cell type that are being induced is the use of morphogens like cytokines or growth factors as additives to the medium. These can be very costly and variable depending on the source.
The method of culturing human pluripotent stem cells can have a significant impact on the ability of those cells to respond to differentiation cues. There are several methods for culturing human pluripotent stem cells including the use of specialized media with (feeder-dependent) or without (feeder-free) co-culture with mouse or human irradiated fibroblasts. Several home-made and commercial media have been developed to promote the maintenance of the pluripotent state in human pluripotent stem cells including KO-DMEM+Knock Out Serum Replacer (KOSR), conditioned medium from irradiated feeder cells, mTeSR™ 1 (STEMCELL Technologies, Inc., Cat #05850, 2008), TeSR™-E8™ (STEMCELL Cat #05840, 2012), Essential-8™ (Life Technologies, Inc., Cat #A14666SA, 2012) and others. Use of each of these specialized media can result in the propagation of human pluripotent stem cells with somewhat different phenotypes. The culture media used to maintain human pluripotent stem cells can therefore affect the ability of these cells to differentiate in response to a given stimulus. For example, human pluripotent stem cells cultured in mTeSR™ 1 are able to respond to the commercially available STEMdiff™ Definitive Endoderm Kit (STEMCELL Technologies, Inc., Cat #05110, 2012) with highly efficient differentiation towards definitive endoderm. Human pluripotent stem cells cultured in TeSR™-E8™ or Essential 8™ are not routinely able to differentiate with high efficiency to definitive endoderm using the STEMdiff™ Definitive Endoderm Kit. Therefore this indicates that there are inherent differences in the functional properties of the stem cells cultured using these different pluripotency or maintenance media which effect their downstream differentiation potential.
Another common approach to control for heterogeneity is the use of selection strategies to obtain the desired cell types, such as mechanical selection or promotion of selective survival using certain media supplements and factors. Mechanical selection can be very tedious and also hardly gives rise to an entirely pure population of desired cell types. A major drawback of using certain supplements for the induction of cell types (e.g. N2 supplement for neural induction) is the interference with cell survival at later stages of the protocol when actual progenitor cells are isolated (Dhara et al., 2008).
Another disadvantage of many protocols is the amount of time it takes to obtain a pure differentiated cell population, especially when the protocol is multi-stepped and includes selection strategies as described above. The entire procedure may take up to several weeks.
Differentiated cell types derived from human pluripotent stem cells are the object of therapeutic approaches such as cell transplantations. Current research aims are focusing on the removal of animal derived proteins from human pluripotent stem cell cultures and differentiated lineages (Mallon et al., 2006). In many current protocols, so-called feeder cells used for the induction of germ layers are commonly derived from mouse tissues. In addition, differentiation of EBs especially into mesoderm often involves the use of fetal bovine serum in culture media, which is a non-characterized animal derived product.
Cell plating densities in monolayer cultures are often not well defined in many protocols and might influence the percentages of the desired cell types induced due to cell intrinsic signaling as described above. Early induction events may also be potentially influenced using differentiation protocols based on EB formation, which deal with variability in EB size and shape.
There is some evidence in the literature that the osmolality of the culture medium influences cell proliferation, survival and differentiation. For example, the osmolality of mTeSR™ 1 medium was adjusted to a higher osmolality of 340 mOsm/kg compared to more standard osmolality of 290-330 mOsm/kg used in most cell culture media to better maintain the undifferentiated state of human ES cells (Ludwig et al., 2006). On the other hand, differentiated cell types such as primary neurons isolated from the CNS survive better in medium with low osmolality (230-280 mOsm/kg) compared to standard osmolality (Brewer et al., 1993; Brewer and Price 1996; Kivell et al., 2000). The available information suggests that a specific osmolality is either effective for maintaining cells in the undifferentiated state, promoting survival or maintaining already differentiated cells or mature cells in the differentiated state.
The lack of standardization of protocols for the differentiation of pluripotent stem cells has also been discussed widely in the literature (for a review see Sanchez-Pernaute and Sonntag, 2006).
Human ES cells are able to give rise to neural tissue in vitro either    1. Directed by an activity inherent to certain embryonic fibroblast cells of murine or human origin. This activity is referred to as stromal derived inducing activity or SDIA (Perrier et al., 2004, Sonntag et al., 2007);    2. Spontaneously under adherent conditions as monolayers as clumps or single cells and with the addition of supplements such as N2 and B27 (Nat et al., 2007) or directed by addition of morphogens (Osafune et al., 2007);    3. Spontaneously as an aggregated mass of differentiating cells known as embryoid bodies (EBs), which is believed to occur because of the presence of inductive factors inside of the EB mimicking the events taking place in the early embryo. These EBs contain cells of all three germ layers, including neuronal cells of the ectodermal lineage (Odorico et al., 2001, Zhang et al., 2001, Yan et al., 2005).
To date, all three of the foregoing methods of inducing neuroectoderm are inefficient and lead to heterogeneous populations of cells, many of which are non-neural.
However, higher efficiencies in neural/neuronal differentiation were achieved when human embryonic stem cells (hESCs) were exposed to morphogens like retinoic acid (Schuldiner et al., 2001), Fgf2 (Zhang et al., 2001), conditioned medium (Schulz et al., 2003; Shin et al., 2006), bone morphogenetic protein (BMP) inhibitors (Itsykson et al., 2005, Gerrard et al., 2005, Sonntag et al., 2007) or SMAD signaling inhibitors such as SB431542 (Chambers et al., 2009; Kim et al., 2010).
Although these protocols increase the emergence of neural cells, a subsequent selection of neural cells from this mixture had to be utilized in a majority of these protocols in order to obtain relatively pure populations of neuronal cells from differentiating cultures of ES cells. In vitro, early emerging neural progenitor cells are morphologically distinct from other cell types and are characterized by the formation of radially organized columnar epithelial cells termed “neural rosettes” (Zhang et al. 2001, 2005; Elkabetz et al., 2008). These structures comprise cells expressing early neuroectodermal markers such as Pax6 and Sox1 and are capable of differentiating into various region-specific neuronal and glial cell types in response to appropriate developmental cues (Yan et al., 2005, Perrier et al. 2004; Li et al. 2005). Over time in culture, the Pax6 positive cells down-regulate Pax6 expression and maintain Sox1 expression. However, they also begin to express Nestin. Similar protein expression profiles are observed in in vivo neural development when comparing neural precursors at the neural plate stage versus neural precursors emerging after neural tube closure (Jessell 2000). Currently, Nestin and Sox1 protein co-expression as well as formation of “neural rosettes” are considered a reliable criterion for the detection of neural progenitor cells (Elkabetz et al., 2008; Elkabetz and Studer 2009; Koch et al., 2009; Peh et al. 2009).
The controlled differentiation of human pluripotent cells into pure or highly enriched population neural progenitor cells and subsequent differentiation of these cells into the 3 cell types of the central nervous system (CNS): neurons, astrocytes and oligodendrocytes without any additional selection procedure would be highly desirable in the field since all these cell populations would provide real advantages for basic and applied studies of CNS development and disease.
To summarize, the field is lacking a standardized media formulation(s) and protocol(s) to induce the 3 germ layers and subsequently more specialized cell types derived thereof in a short period of time. The field also suffers from the lack of standardized protocols which are easy to reproduce in different labs and operator-independent. Furthermore, the field suffers from a lack of formulation(s) and protocol(s) that allow for efficient differentiation to the 3 germ layers or to a specific germ layer from pluripotent stem cells which are cultured under varying maintenance or pluripotency culture conditions.